Abstract
Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S). In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as well as after its removal. Virginiamycin M inhibited the binding of puromycin to ribosomes and reduced both the enzymatic and nonenzymatic binding of Phe-tRNA to the A site by inducing its release from the ribosomes (similar effects were observed with 50 S), whereas the antibiotic had no effect on the binding of unacylated tRNAPhe to the same site. Moreover, virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP (similar finding with 50 S). Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic. The overall conclusion is that both the acceptor and donor substrate binding sites of the peptidyltransferase, which interact with the aminoacyl moiety of tRNA, are permanently altered upon transient contact of ribosomes with virginiamycin M.
Highlights
Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S*).In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as wellas after itsremoval
Virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP.Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic
In agreement with these earlier observations, we found that ["C] tRNAPhebound to the P site of poly(U)-ribosome complexes did not exchange with free unlabeled tRNA nor was it released from ribosomes by EF-G plus GTP in the presence of 14 mM M P. When this experiment was repeated with ribosomal complexes carrying Ac-[14C]Phe-tRNAPhaet theP site, extensive release of this tRNAspecies was observed as well in the presence of virginiamycin M as with 70 S* particles (Fig. 4 B ), but not in the absence of the antibiotic (Fig. 4A). These results indicate that thestability of the linkage of AcPhe-tRNA with the P site is due to an interaction of the acetyl-aminoacyl moiety of this substratewith the donor site of the peptidyltransferase; virginiamycin M causes a permanent alteration of this site, producing a release of the otherwise stably bound Ac-Phe-tRNA
Summary
Ribosomes andProteinSynthesis Factors-Escherichia coli A19 cefls were disrupted either by grinding with alumina or by a French press operated a t 12,000 p.s.i. Sucrose-washed ribosomes from cells disrupted by the French press were used to study the effect of virginiamycin M on the binding of [3H]puromycinto ribosome by equilibrium dialysis These ribosomes showed a high affinity for puromycin and were essentially free of peptidyl-tRNA (less than 0.003 mol of puromycin/mol of ribosomes incorporated into cold trichloroacetic acid-insoluble material). Total E. coli tRNA synthetases obtained as Formation of Ribosomal Complexes Carrying Ac-p4C]Phe-tRNAPk described by Muench and Berg [29] were used for preparative ami- in the P Site-70 S and 70 S*particles (13A2m units of ribosomes in noacylation of tRNAPhe. 150 pl of standard buffer with 7 mM MgCIz)were incubated 20 min Nucleotdes and Nucleic Acids-ATP, GTP, poly(U), and tRNA a t 30 "C with 25 pg of poly(U) and 100 pmol of Ac-["CIPhe-tRNA.
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